Cyclosporine derivatives and uses thereof

ABSTRACT

The present invention provides novel cyclosporine C (CsC) derivatives having improved protein conjugatibility and hydrolytic stability. The present invention further provides a CsC derivative conjugated to a carrier, e.g., a solid support. Preferably, the solid support is a latex or magnetic particle.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The present invention relates to novel cyclosporine derivatives thathave improved protein and solid surface conjugatibility and hydrolyticstability. The cyclosporine derivatives of the present invention areuseful in assays measurement of cyclosporin A levels, as well as in theproduction of cyclosporine immunogens and capture conjugates.

2. Background

Cyclosporine A (cyclosporine) is a potent immuno-suppressant that hasbeen widely used in the United States and other countries to prevent therejection of transplanted organs such as kidney, heart, bone marrow andliver, in humans.

To prevent allograft rejections, minimum level cyclosporine A in theblood is required throughout the lifetime of the patient. Chronic highdoses can result in kidney and liver damage. Distribution and metabolismof the drug varies greatly between individuals, as well as in a singleindividual during the course of therapy. Accordingly, monitoringcyclosporine A levels in the blood or serum of allograph recipients isconsidered essential.

Laboratory methods for detection of cyclosporine have been developed.These techniques typically involve high performance liquidchromatography (HPLC), radioimmunoassay (RIA) and non-radioimmunoassay.

It has been reported that CsA, itself, is non-immunogenic (Donatsch, p.et al., J. Immuno Assay 1981; 2:19). To obtain antibodies, therefore, itis necessary to link CsA to a protein carrier. The side chain of CsA,however, consists most of alliphatic groups. Few of the functionalgroups customarily used to link a hapten to a carrier. Previous workershave made immunogenic cyclosporine C (CsC) protein conjugates becausethe CsC has a threonine residue in position 2. Linkage to a protein wasvia a hemisuccinate linker through an ester group (U.S. Pat. No.5,169,773). In addition, hemisuccinate coupling chemistry has been usedto immobilize CsC to a solid support such as stabilized chromium dioxideparticles (U.S. Pat. No. 5,151,348). Due to a number of factorsincluding, for example, short chain length of the hemisuccinate linker,hydrophobicity of the cyclosporine-C hemisuccinate molecule andhydrolytic instability of the hemisuccinate ester linkage, the CsChemisuccinate derivatives conjugate poorly to protein and solid surface.Furthermore, CsC protein conjugates and CsC immobilized on a solidsupport by hemisuccinate coupling, are hydrolyticly unstable. Thus,immunoassays developed by using such hemisuccinate CsC derivativessuffer from low sensitivity and poor reagent stability. There is astrong desire to replace the widely used radioimmunoassays and HPLCmethods with a more robust and sensitive immunoassay for CsA.Accordingly, there is a need in the art for cyclosporine derivativesthat are capable of being conjugated to solid supports and carriers moreefficiently and stably.

SUMMARY OF THE INVENTION

The present invention provides novel cyclosporine C (CsC) derivativeshaving improved protein conjugatibility and hydrolytic stability. Thepresent invention further provides a CsC derivative conjugated to acarrier, e.g., a solid support. Preferably, the solid support is a latexor magnetic particle.

The invention also provides improvements in assays for the determinationof cyclosporin levels in a sample, e.g., whole blood, suspected ofcontaining cyclosporin.

Furthermore, the invention includes kits for conducting an assay for thedetermination of cyclosporin. The present invention also provides forthe production of cyclosporine immunogens and capture conjugatescomprising the CsC derivatives of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to cyclosporine C (CsC) derivatives havingthe structure: ##STR1## x is selected from the group consisting of:##STR2## Y is selected from the group consisting of: ##STR3## wherein Zis selected from the group consisting of; ##STR4## wherein R₁ and R₂ areeach a C1-C8 alkyl group; wherein R₃ is a C₀ -C₈ alkyl group; and

wherein m is 1-200

A particular embodiment of the present invention includes CsCderivatives having the following structures: ##STR5##

The CsC derivatives of the present invention were prepared by activationof the CsC position 2 hydroxy group using disuccinimidyl carbonatefollowed by coupling with linkers such as diamine linkers, e.g.,ethylene glycol bis(2-aminoethyl)ether (DA- 10). The following schemeillustrates the application of this procedure to the synthesis of theCsC derivatives of this invention: ##STR6##

Starting materials used in the above-described scheme are either knownor commercially available.

The CsC derivatives of the present invention may be used in animmunoassay for the measurement of cyclosporin A levels in whole bloodsamples. An example of such an assay comprises the steps of:

(a) lysing red blood cells in a sample of whole blood containingcyclosporin A;

(b) contacting the lysed whole blood sample with excess labeledanti-cyclosporin antibody, e.g., beta -D-galactosidase labeled, to forma labeled antibody-cyclosporin A complex;

(c) separating unbound antibody from the complex by contacting themixture formed in step (b) with a solid phase comprising a cyclosporinderivative of the present invention immobilized on a solid support; and

(d) determining the amount of the label in the complex as a measure ofcyclosporin A, using, for example, a beta -D-galactosidase substrateselected from the group consisting of chlorophenol red- beta-D-galactopyranoside (CPRG) and resorufin- beta -D-galactopyranoside(ReG) if a beta -D-galactosidase label is used.

The enzyme-linked immunoassay of this invention is useful for measuringcyclosporin A levels in whole blood samples of patients receivingcyclosporin A. Monitoring of cyclosporin A blood levels and subsequentcyclosporin A dosage adjustment are necessary to prevent toxic effectscaused by high cyclosporin A blood levels and to prevent organ rejectioncaused by low cyclosporin A blood levels.

The immunoassay of the present invention is performed by contacting alysed whole blood sample containing cyclosporin A with excess labeledanti-cyclosporin antibody, e.g., beta -D-galactosidase-labeled, to forma reaction mixture containing a complex of cyclosporin A with labeledantibody and free labeled antibody, separating free antibody from thereaction mixture by contacting the reaction mixture with a solid phasecomprising an immobilized CsC derivative of the present invention on asolid support, e.g. magnetic particles, separating the solid phase fromthe liquid phase, and measuring the amount of the bound label in theliquid phase by adding, for example, to the liquid phase CPRG or ReG asa beta -D-galactosidase substrate if a beta -D-galactosidase label isused.

Specifically, the red blood cells of a whole blood sample containingcyclosporin A must be lysed to release cyclosporin A. Red blood celllysis can be accomplished by many methods, such as sonication, detergentlysis and distilled water lysis. The lytic agent chosen should becompatible with the labeled anti-cyclosporin antibody. Although somedetergents can denature beta -D-galactosidase, it has been found that byusing CPRG and ReG as beta -D-galactosidase substrates, the samplevolume can be made to be sufficiently small to minimize the denaturingeffect of the detergent. The preferred lysis method uses detergent.

After lysis, a reaction mixture is formed by contacting the lysed wholeblood sample with excess labeled anti-cyclosporin antibody andincubating the reaction mixture for a time and at a temperaturesufficient to permit the labeled antibody to form a complex with all ofthe cyclosporin A in the sample. This usually takes 1-5 minutes at roomtemperature. Anti-cyclosporin antibody can be obtained commercially,prepared by known methods, or prepared using the derivatives of thepresent invention. The anti-cyclosporin antibody can be polyclonal ormonoclonal. A monoclonal anti-cyclosporin antibody specific forcyclosporin A is preferred. The anti-cyclosporin antibody can be labeledusing standard techniques with any molecule that can be detected,including, for example, radioactive isotopes, a catalyst such as anenzyme (e.g., beta -D-galactosidase), a co-enzyme, a chromogen such as afluorescer, dye or chemiluminescer, a dispersible particle that can benon-magnetic or magnetic, a solid support, a liposome, a ligand, ahapten, and so forth.

The unbound anti-cyclosporin antibody is separated from the reactionmixture by contacting the reaction mixture with a solid phase comprisinga CsC derivative of the present invention immobilized on a solid supportfor a time sufficient to permit the unbound labeled antibody to form acomplex with the immobilized CsC derivative. This usually occurs inapproximately one minute.

The immobilization of the CsC derivative of the present invention can beaccomplished by a number of known immobilization techniques. Thepreferred immobilization technique for derivatives of the presentinvention is to activate the terminal carboxy group, using for example,2-Fluoro-lmethylpyridinium p-toluenesulfonate (FMPT), and then couplingto a protein, such as albumin or globulin, which can be covalentlycoupled to a solid support.

The CsC derivative can be immobilized on a variety of solid supportssuch as beaded dextran, beaded agarose, polyacrylamide, or glass. Apreferred solid support useful in the immunoassay of this invention isdescribed in U.S. Pat. No. 5,151,348 and 5,302,532.

The preferred solid support comprises a stabilized chromium dioxideparticles having cyclosporin bound to their surfaces. The stabilizedchromium dioxide particle useful in the preferred solid support arethose described in U.S. Pat. No. 4,661,408. These particles consist of acore of rutile chromium dioxide which has been extensively surfacereduced, coated with alumina, further coated with silica containingborate and still further coated with a silane to which is attachedcyclosporin protein conjugate, such as bovine gamma globulin. Theseparticles have large surface areas, 40-100 m² /g, are stable in aqueoussolution and can be readily coupled to cyclosporin conjugate.

The support can also be a porous or non-porous water insoluble material.The support can be hydrophilic or capable of being rendered hydrophilicand includes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly(vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass, ceramics, metals, and the like.

The solid phase is separated from the liquid phase by standardseparation techniques. The preferred separation technique is to usemagnet to settle the magnetic particle out of liquid phase.

The amount of cyclosporin A is determined by measuring the amount of thebound label in the liquid phase. For example, the amount of bound beta-D-galactosidase is determined by adding to the liquid phase either CPRGor ReG as a beta -D-galactosidase substrate and measuringspectrophotometrically the amount of chromophore produced at 577 nm.

The immunoassay of this invention can be performed manually or it can beadapted to a variety of automated or semi-automated instrumentation,such as the Dimension ® RxL (discrete clinical analyzer, a registeredtrademark of Dade International Inc. Deerfield, Ill.). In performing theassay on a Dimension ® RxL, a whole blood sample is first lysed andpreincubated with excess beta -D-galactosidase-labeled anti-cyclosporinantibody in a cuvette on the instrument. A known amount of stabilizedchromium dioxide particle immobilized with CsC derivative of presentinvention is transferred into the cuvette and incubated for certainamount time, use magnet to separate the magnetic particle from liquidphase. The liquid phase of supernatant contains beta-D-galactosidase-labeled anti-cyclosporin antibody complexed withcyclosporin A from the whole blood sample. A fraction of the supernatantis pipetted and transferred to another cuvette with the addition of CPRGor ReG immediately preceding the absorbance measurements at 577 nm.

The present invention further provides a CsC derivative conjugated to acarrier, which is generally a compound of molecular weight greater than5,000, or a label. Carriers include polyamino acids,lipopolysaccharides, and particles. The CsC conjugate can be used inmany applications including as a capture conjugate in an assay or as animmunogen. The carrier may be immunogenic, i.e,. an immunogenic carrier.

The poly(amino acids) will generally range from about 5,000 molecularweight, having no upper molecular weight limit, normally being less than10,000,000, usually not more than about 600,000 daltons.

Various protein types may be employed as the poly(amino acid)immunogenic material. These types include albumins, serum proteins,e.g., globulins, ocular lens proteins, lipoproteins, etc. Illustrativeproteins include bovine serum albumin, keyhole limpet hemocyanin, eggovalbumin, bovine gamma -globulin, etc. Alternatively, syntheticpoly(amnino acids) may be utilized.

The immunogenic carrier can also be a polysaccharide, which is a highmolecular weight polymer built up by repeated condensations ofmonosaccharides. Examples of polysaccharides are starches, glycogen,cellulose, carbohydrate gums, such as gum arabic, agar, and so forth.The polysaccharide can also contain polyamino acid residues and/or lipidresidues.

The immunogenic carrier can also be a nucleic acid either alone orconjugated to one of the above mentioned poly(amino acids) orpolysaccharides.

The carrier can also be a particle. The particles are generally at leastabout 0.02 microns and not more than about 100 microns, usually at leastabout 0.05 microns and less than about 20 microns, preferably from about0.3 to 10 microns diameter. The particle may be organic or inorganic,swellable or non-swellable, porous or non-porous, preferably of adensity approximating water, generally from about 0.7 to about 1.5 g/ml,and composed of material that can be transparent, partially transparent,or opaque. The particles can be biologic materials such as cells andmicroorganisms, e.g., erythrocytes, leukocytes, lymphocytes, hybridomas,streptococcus, staphylococcus aureus, E. coli, viruses, and the like.The particles can also be particles comprised of organic and inorganicpolymers, liposomes, latex particles, phospholipid vesicles,chylomicrons, lipoproteins, chrome and the like.

The particles can be derived from naturally occurring materials,naturally occurring materials which are synthetically modified andsynthetic materials. Among organic polymers of particular interest arepolysaccharides, particularly cross-linked polysaccharides, such aagarose, which is available as Sepharose, dextran, available as Sephadexand Sephacryl, cellulose, starch, and the like; addition polymers, suchas polystyrene, polyvinyl alcohol, homopolymers and copolymers ofderivatives of acrylate and methacrylate, particularly esters and amideshaving free hydroxyl functionalities, and the like.

The particles will usually be polyfunctional and will be bound to or becapable of binding to the CsC derivative. A wide variety of functionalgroups are available or can be incorporated. Functional groups includecarboxylic acids, aldehydes, amino groups, cyano groups, ethylenegroups, hydroxyl groups, mercapto groups and the like. The manner oflinking a wide variety of compounds to particles is well known and isamply illustrated in the literature. See for example Cautrecasas, J.Biol. Chem. (1970) 245:3059.

The carrier can be an enzyme that is part of a signal producing system.The function of the signal producing system is to produce a productwhich provides a detectable signal related to the amount of bound andunbound label. Where enzymes are employed, the involved reactions willbe, for the most part, hydrolysis or redox reactions. Such enzymes thatmay find use are hydrolases, transferases, lyases, isomerases, ligasesor synthetases and oxidoreductases, preferably hydrolases.Alternatively, luciferases may be used such as firefly luciferase andbacterial luciferase.

A label may be any molecule conjugated to an analyte or an antibody, orto another molecule. In the subject invention, the label can be a memberof the signal producing system that includes a signal producing means.The label may be isotopic or nonisotopic, preferably nonisotopic. By wayof example and not limitation, the label can be a catalyst such as anenzyme, a co-enzyme, a chromogen such as a fluorescer, dye orchemiluminescer, a dispersible particle that can be non-magnetic ormagnetic, a solid support, a liposome, a ligand, a hapten, and so forth.

The signal producing system may have one or more components, at leastone component being a label. The signal producing system includes all ofthe reagents required to produce a measurable signal including signalproducing means capable of interacting with the label to produce asignal.

The signal producing system provides a signal detectable by externalmeans, normally by measurement of electromagnetic radiation, desirablyby visual examination. For the most part, the signal producing systemincludes a chromophoric substrate and enzyme, where chromophoricsubstrates are enzymatically converted to dyes which absorb light in theultraviolet or visible region, phosphors or fluorescers.

The signal producing means is capable of interacting with the label toproduce a detectable signal. Such means include, for example,electromagnetic radiation, heat, chemical reagents, and the like. Wherechemical reagents are employed, some of the chemical reagents can beincluded as part of a developer solution. The chemical reagents caninclude substrates, coenzymes, enhancers, second enzymes, activators,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Some of the chemical reagents such as coenzymes, substances thatreact with enzymic products, other enzymes and catalysts, and the likecan be bound to other molecules or to a support.

The signal producing system including the label can include one or moreparticles, which are insoluble particles of at least about 50 nm and notmore than about 50 microns, usually at least about 100 nm and less thanabout 25 microns, preferably from about 0.2 to 5 microns, diameter. Theparticle may be organic or inorganic, porous or non-porous, preferablyof a density approximating water, generally from about 0.7 to about 1.5g/ml, and composed of material that can be transparent, partiallytransparent, or opaque.

The label can also be fluorescent either directly or by virtue offluorescent compounds or fluorescers bound to a particle in conventionalways. The fluorescers will usually be capable of, or functionalized torender them capable of, being bound to the CsC derivative or to theparticle.

The CsC derivatives of the present invention can be utilized to prepareconjugates using the reactions discussed above and set forth in theexamples.

Another aspect of the present invention includes antibodies prepared inresponse to a CsC derivative conjugated to an immunogenic carrier.Furthermore, the present invention includes conjugates of suchantibodies and a label.

An antibody is an immunoglobulin which specifically binds to and isthereby defied as complementary with a particular spatial and polarorganization of another molecule. The antibody can be monoclonal orpolyclonal and can be prepared by techniques that are well known in theart such as immunization of a host and collection of sera from which theimmunoglobulin can be separated by known techniques (polyclonal) or bypreparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal). Antibodies may include a complete immunoglobulinor fragment thereof, which immunoglobulins include the various classesand isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM,etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and thelike.

Monoclonal antibodies can be obtained by the process discussed byMilstein and Kohler and reported in Nature, 256:495-497, 1975.

The antibodies of the present invention recognize the cyclosporinsincluding cyclosporin A and derivatives and metabolites of cyclosporins.

The antibodies of the present invention can be utilized in thedetermination of cyclosporin in a sample suspected of containingcyclosporin. The assay can comprise the steps of contacting the samplewith antibodies for cyclosporin and detecting either directly orindirectly immune complexes of the antibodies and cyclosporin. Theimprovement provided in the present invention is the utilization of thepresent antibodies as the antibodies for cyclosporin. The immunecomplexes are detected directly, for example, where the antibodiesemployed are conjugated to a label. The immune complex is detectedindirectly by examining for the effect of immune complex formation in anassay medium, on a signal producing system or by employing a labeledantibody that specifically binds to an antibody of the invention.

In another configuration of an assay for the determination ofcyclosporin in a sample suspected of containing cyclosporin, the sampleis contacted with antibodies for cyclosporin and a conjugate of thisinvention recognized by the antibodies. The method further includesdetecting either directly or indirectly immune complexes of theconjugate and the antibodies. The improvement provided in the presentinvention is employing as the CsC derivative conjugated to a label.

The present assay invention has application to all immunoassays forcyclosporin. The assay can be performed either without separation(homogeneous) or with separation (heterogeneous) of any of the assaycomponents or products. Exemplary of heterogeneous assays are enzymelinked immunoassays such as the enzyme linked immunosorbant assay(ELISA), see "Enzyme-Immunoassay" by Edward T. Maggio, CRC PressIncorporated, Boca Raton, Fla., 1980. Homogeneous immunoassays areexemplified by enzyme multiplied immunoassay techniques (e.g. see U.S.Pat. No. 3,817,837), immunofluorescence methods such as those disclosedin U.S. Pat. No. 3,993,345, enzyme channeling techniques such as thosedisclosed in U.S. Pat. No. 4,233,402, and other enzyme immunoassays asdiscussed in Maggio, supra.

The references cited throughout the specification are hereinincorporated by reference.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

EXAMPLE 1 Synthesis of CsA-DA-10

In a 25 mL round bottom flask equipped with magnetic stirrer, 540 mg ofCsC and 454 mg of DSC (disuccinimidyl carbonate) was placed, then 10 mLof dry acetonitrile and 1000 μL of triethyl amine were added. Thereaction was stirred at room temperature for about 16-20 hours until noCsC could be found on TLC (thin layer chromatography). If the reactionwas not complete after 16-20 hours, 50 mg more of DSC were added intothe solution and the reaction checked again after 2 hours by TLC. (95%EtOAc and 5% MeOH were used as TLC solvent, iodine was used to visualizethe spots.). To the solution was then added 2627 mg of DA-10 (quickly)and 1000 μL of triethyl amine. The reaction was stirred at roomtemperature for another 24 hours, and then 50 mL of CH₂ CI₂ added. Thereaction solution was washed with water 3 times. The bottom organiclayer was separated and dried with sodium sulfate. The white solid ofCsA-DA-10 product was obtained after removal of all solvent by rotaryevaperation and vacuum.

EXAMPLES 2 Synthesis of CsA-DA- 10-HemiGlutarnate (HG)

In a 20 mL vial, about 550 mg of CsA-DA-10, 93.75 mg of glutaricanhydride and 10 mL CH₂ CI₂ were placed; 344 μL of triethyl amine wasadded and the reaction solution stirred at room temperature for about 2hours. To the reaction solution was then added 40 mL of CH₂ CI₂, andwashed with 1N HC1 twice and water twice. The organic layer was driedwith sodium sulfate, and solid CsA-DA- 10-HG was obtained after removalof all solvent.

EXAMPLE 3 Conjugation of CsA-DA-10-HG with Bovine Gamma Globulin (IgG)

A: 4 mg/mL of Bovine gamma globulin Solution: 500 mg of IgG dissolvedinto 125 mL of 0.1M Na₂ CO₃ (pH 9.5).

B: Activation of CsA-DA-10-HG with 2-Fluoro- Imethylpyridiniump-toluenesulfonate (FM PT).

134.6 mg of CsA-DA-10-HG (0.0894 mmol) and 38.1 mg of FMPT (0.1345mmole) were weighed. 4800 μL of dried CH₃ CN to dissolve the solids wasadded. 28.1 μL of TEA was then added. The reaction was stirred at roomtemperature for 2 hr.

C: Coupling of CsA-DA-10-HG to Protein

The above activated CsA-DA-10-HG solution was added to the 4 mg/mL ofBovine gamma globulin solution with stirring, allowing each drop todisperse before the next one was added. After the addition was complete,the solution was allowed to stir gently for about 18 hours at roomtemperature. The solution was dialyzed (12,000 MW cut-off dialysis tube)in a cold room against 6 changes of PBS buffer solution. The dialyzedsolution was diluted to 2 mg/mL of the protein concentration by addingfresh dialysis PBS buffer. 324 mg of brontopol was added to thecontainer and agitated until it completely dissolved and was uniformlydispersed.

EXAMPLE 4 Coupling CsA-Bovine Gamma Globulin Conjugate to A MagneticParticle

40 ml of above CsA-IgG conjugate was added into 40 ml of glutaraldhydeactivated chromium dioxide solution. (The detailed procedure ofpreparation of activated chromium dioxide particle is disclosed in U.S.Pat. No. 4,661,408, the disclosure of which is incorporated herein byreference). The reaction was rotated at 4° C. for 24 hours. 32 ml of 30%BSA was added into the solution, the reaction rotated at roomtemperature for another 16-20 hours. 112 ml of 2 M glycine buffer wasadded into the above solution to quench the reaction for about 1 hour.The solution was washed with water three times and chrome diluent threetimes. The chromium dioxide particle was diluated to 40 ml with chromediluent.

    ______________________________________                                        Chrome diluent:                                                               ______________________________________                                        Polymerized BSA (30%)                                                                           15.1       g/L                                              Treholose         28.7       g/L                                              Carbowax          4.8        g/L                                              Proclin           5          mL/L                                             Nemycin Sulfate   0.06       g/L                                              ______________________________________                                    

This invention has been described in detail including the preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements thereon without departing from the spiritand scope of the invention as set forth in the claims.

What is claimed is:
 1. A compound having the structure: ##STR7## whereinX is selected from the group consisting of: ##STR8## wherein Y isselected from the group consisting of: ##STR9## wherein Z is selectedfrom the group consisting of: ##STR10## wherein R₁ and R₂ are each a C₁-C₈ alkyl group; wherein R₃ is a bond or a C₁ -C₈ alkyl group;andwherein m is 1-200.
 2. A compound having the structure: ##STR11##wherein n is 2-; m is 2-5 and p is 2-3.
 3. A compound having thestructure: ##STR12## wherein n is 2-3; m is 2-5; p is 2-3 and k is 2-3.